Method of selective isolation or visualization of target cells differentiated from embryonic cells or kit for visualization

ABSTRACT

A method for selectively isolating or visualizing a target cell differentiated from an embryonic stem cell, which comprises transferring a first recombinant DNA in which a first promoter, a gene having recombinase recognition sequences on both ends, and a selective marker gene of a target cell differentiated from an embryonic stem cell are arranged in this order from a 5′ side, and the first promoter makes the selective marker gene express, and a second recombinant DNA in which a second promoter specifically expressing in a target cell differentiated from an embryonic stem cell, and a recombinase-expressing gene are arranged in this order from a 5′ side, respectively into an embryonic stem cell. A kit for isolation or visualization, which comprises the first vector for transferring a gene containing a first DNA and the second vector for transferring a gene containing a second DNA.

TECHNICAL FIELD

The present invention relates to a method which can effectively andassuredly select and isolate, or visualize target cells differentiatedembryonic stem cells and a kit for isolation or visualization usedtherefor.

BACKGROUND FIELD

An embryonic stem (ES) cell (hereinafter, also referred to as ES cell)is a cell separated from a primary embryo, and this has such totipotencethat the cell can be differentiated into any organ or cell such ashemocyte, cardiac muscle, skeletal muscle and nerve ones by manipulatingthe culturing system, development of study in the field of biology suchas embryology, and advanced medicine utilizing this is greatly expected.For such the study, establishment of a method which effectively andassuredly selects and isolates target cells differentiated from an EScell is one of the most important themes.

Meanwhile, when differentiated target cells express a specific membraneprotein hitherto, target cells have been isolated by flow cytometryusing the membrane protein as an index (Yohei Morita, Multiple StainingAnalysis for Lymphocyte Subset: supervised by Hiromitsu Nakauchi, editedby Yayoi Tanaka, Freely Performed Flow Cytometry (Cell TechnologySupplement), Sujunsya, p60-66(1999)/Yamashita J, Itoh H, Hirashima M,Ogawa M, Nishikawa S, Yurugi T, Naito M, Nakao K, Nishikawa S.Flk1-positive cells derived from embryonic stem cells serve as vascularprogenitors. Nature 408(6808): p92-6(2000)).

However, since application of this method can be limited to the casewhere a cell-specific molecule is expressed extracellularly as amembrane protein, application is limited, in fact, to a part of cellsand organs such as hemocyte system and vascular system.

For this reason, as strategy for many cells for which a specificmembrane protein is not known such as a cardiac muscular cell, there isreported a method of stably transferring into an ES cell a recombinantgene in which a marker gene is ligated under a promoter region of amolecule (gene) expressed specifically in a target cell, and selectingand isolating a cell using a marker expressed specifically in adifferentiated cell as an index (Andressen C, Stocker E, Klinz F J,Lenka N, Hescheler J, Fleischmann B, Arnhold S, Addicks K.Nestin-specific green fluorescent protein expression in embryonic stemcell-derived neural precursor cells used for transplantation. StemCells19(5):419-24(2001)). This method is a method of using a promoter of agene expressed specifically in a tissue to express a drug-resistant genetissue-specifically, and selecting only a target cell using the drug, ora method of selecting and isolating a target cell by flow cytometry byexpressing cell-specifically a molecule which develops a color withexcitation light having a specific wavelength.

However, these methods have a great problem that they remarkably dependon activity (expression intensity) of a cell-specific promoter, andtheir application and efficacy are considerably limited. That is, when acell-specific promoter has, if any, weak specificity, since sufficientintensity of expression of a marker gene for selecting and isolating adifferentiated target cell can not be obtained, and a target cell cannot be selected, this method has, in fact, considerably limitedapplication. In addition, it is difficult to predict whether this methodis assuredly useful or not at a stage of planning an experiment, andpredicted result is not certain. Nevertheless, for differentiation intoa target cell and isolation of the cell, an ES cell strain must beprepared one by one every purpose, and a differentiated target cell mustbe tested one by one, therefore, a target cell can not be consequentlyisolated in some cases although much labor and time are consumed.

By allowing even a tissue-specific promoter having low activity(expression intensity) to be expressed at a sufficient amount in orderto have a marker gene function, the present invention provides a methodfor assuredly, simply and rapidly selecting and isolating adifferentiated target cell which can be utilized in various study fieldssuch as medicine, biology and biotechnology as well as regenerationtherapy using ES cells of various animals, a method for selectivelyvisualizing such the target cell, and a kit for isolation andvisualization which is used therefor.

DISCLOSURE OF THE INVENTION

The present invention is a method for selectively isolating orvisualizing a target cell differentiated from an embryonic stem cell,which comprises transferring a first recombinant DNA in which a firstpromoter, a gene having recombinase-recognition sequences on both ends,and a selective marker gene for a target cell differentiated from anembryonic stem cell are arranged in this order from a 5′ side, and thefirst promoter makes the selective marker gene to be expressed, and asecond recombinant DNA in which a second promoter specificallyexpressing a target cell differentiated cell embryonic stem cell, and arecombinase-expressing gene are arranged in this order from a 5′ side,into an embryonic stem cell, respectively. In the aforementionedinvention, transfer of the first recombinant DNA or the secondrecombinant DNA into an embryonic stem cell may be performed using avector for transferring a gene. As this vector for transferring a gene,an adenovirus vector may be used.

In addition, the present invention is an embryonic stem cell in which afirst recombinant DNA and a second recombinant DNA of the aforementionedinvention are transferred.

In addition, the present invention relates to a vector for transferringa first gene containing the aforementioned first recombinant DNA or avector for transferring a second gene containing the aforementionedsecond recombinant DNA.

In addition, the present invention relates to a kit for isolation orvisualization used for a method for selectively isolating or visualizinga target cell differentiated from an embryonic stem cell, whichcomprises a first vector for transferring a gene containing theaforementioned first recombinant DNA, and a second vector fortransferring a gene containing the aforementioned second recombinantDNA.

In addition, the present invention relates to a kit for isolation orvisualization used in a method for selectively isolating or visualizinga target cell differentiated from an embryonic cell, which comprises anembryonic stem cell in which the aforementioned first recombinant DNA istransferred, and a second vector for transferring a gene containing theaforementioned second recombinant DNA.

In addition, the present invention relates to a kit for isolation orvisualization used in a method for selectively isolating or visualizinga target cell differentiated from an embryonic stem cell, whichcomprises a first vector for transferring a gene containing theaforementioned first recombinant DNA, and an embryonic stem cell inwhich the aforementioned second recombinant DNA is transferred.

In addition, the present invention relates to a cell obtained by theaforementioned method for selectively isolating a target celldifferentiated from an embryonic stem cell or a tissue containing thiscell.

In addition, the present invention relates to a method for treating adisease using the aforementioned cell and/or tissue.

The method for selectively isolating or visualizing a target celldifferentiated from an embryonic stem cell of the present invention canbe applied to ES cells derived from various animals. For example, themethod can be used not only already established species such as mouse,rat, monkey and human, but also other species ES cells which will beestablished from now on, and is not particularly limited by an animalspecies and a kind of ES cells.

A standard method for handling ES cells is described in “Manual ofManipulation of Mouse Embryo” authored by Brigid Hogan and others,translated by Kazuya Yamauchi and others, Kindai Publisher (1997), or“Gene Targeting: Preparation of Mutated Mouse Using ES Cells” authoredby Shinichi Aizawa, Experimental Medicine Supplement, Biomanual Series8, Yodosha (1995).

The first recombinant DNA used in the present invention is a DNA inwhich a first promoter, a gene having recombinase-recognition sequenceson both ends, and a selective marker gene for a target celldifferentiated from an embryonic stem cell are arranged in this orderfrom 5′, which has been made by the gene recombination technique. Thefirst promoter is not particularly limited as far as it is a promoterwhich has higher activity than that of a second promoter being notcapable of sufficiently expressing a selective marker gene, and issufficient for expressing a selective marker gene. For example, aconstitutive strong expression promoter such as a CA (hybrid promoter ofcytomegalovirus enhancer and chicken β actin promoter) promoter and aCMV (cytomegalovirus early gene enhancer•promoter) promoter is suitable.The constitutive strong expression promoter refers to a promoter whichhas a target gene to be expressed constitutively and strongly when atarget gene ligated to the promoter is transferred into most cellsincluding an ES cell.

The recombinase recognition sequence is not particularly limited as faras it is a base sequence recognized by recombinase which is a specificDNA recombinant enzyme, and refers to a specific base sequence such asloxP and FRT which causes a DNA recombination reaction such as cleavage,substitution and binding of a DNA chain held by two recombinaserecognition sequences by recombinase.

The recombinase-expressing gene is a gene for expressing recombinase,and representative examples include genes expressing recombinase Crederived from bacteriophage P1 recognizing loxP (Sternberg et al., J.Mol. Boil. Vol. 150, 467-486(1981)), recombinase FLP derived fromSaccharomyces cerevisiae recognizing FRT (Babineau et al., J. Biol.Chem. Vol. 260, 12313-12319 (1985)), and R derived from pSR1 plasmid ofZygosaccharomyces ruii (Matsuzaki et al., Mol. Cell. Biol. Vol. 8,955-962 (1988)), being not limiting.

The selective marker gene is used as an index for specifically selectinga target cell expressed by a first promoter and differentiated from anES cell after a second recombinant DNA is transferred into an ES cell,and examples thereof include an light emitting protein gene such as EGFP(Enhanced Green Fluorescent Protein) and GFP (Green FluorescentProtein), and various drug-resistant genes, being not limited to them asfar as the gene is used as a selective marker. In particular, a lightemitting protein gene is more preferable because it can visualize atarget cell, and selective isolation becomes easy by using flowcytometry. In addition, since a light emitting protein gene canpermanently and constitutively label and visualize also a cell which hasfurther been differentiated from a differentiated target cell when aconstitutively strong expression promoter is used, it becomes possibleto observe and analyze a differentiation line and a tissue line on aculturing dish, being preferable.

The second recombinant DNA used in the present invention is a DNA inwhich a second promoter for specifically expressing a target celldifferentiated from an embryonic stem cell, and a recombinase-expressinggene are arranged in this order from 5′, which has been made by the generecombination technique. The second promoter refers to a promoter regionof a gene which is specifically expressed only in a differentiatingtarget cell. Examples include promoters of genes such as Nkx2.5, MEF-2,GATA-4, cardiac muscle-type actin, α-cardiac myosin heavy chain(hereinafter, αMHC) protein, and myosin light chain-2v (MLC2v) proteinof cardiac muscle cell, nestin of brain nerve cell, glial fibrillaryacidic protein (GFAP) of brain glial cell, α-fetroprotein (AFP) of moreundifferentiated hepatocyte, albumin of (mature) hepatocyte, osteocalcinof osteoblast, pancreatic and duodenal homeobox gene 1 (PDX-1) ofpancreatic β cell, flt-1 of blood vessel (endothelial cell), keratin 14(K14) of an epidermal keratin cell, and muscle creatine kinase ofskeletal muscle cell. Recombinase-expressing genes ligated to thesepromoters express recombinase only when an ES cell is differentiatedinto the target cell.

For transferring the first recombinant DNA or the second recombinant DNAinto an ES cell, a plasmid having a drug-resistant gene together witheach recombinant DNA is transferred by a general method of molecularbiology such as an electroporation method, a calcium phosphate method, aliposome method and a DAE dextran method and, thereafter, a cell iscultured in a medium with a drug added for 1 to 2 weeks, thereby, aclone of an ES cell in which each of these recombinant DNAs isincorporated into a chromosome and which expresses a gene stably andpermanently is collected, and can be used in an experiment fordifferentiation. As a further useful method, each vector fortransferring a gene containing the first recombinant DNA or the secondrecombinant DNA is prepared, and a recombinant DNA can be transferredinto an ES cell simply and very effectively by this. Examples of avector for transferring a gene include an adenovirus vector, aretrovirus vector, a lentivirus vector, an adeno-associated vector, anda Sendaivirus vector, as well as a cationic liposome and a HVJ liposomeas a none-virus vector, being not limiting.

Then, principle of the method for selectively isolating or visualizing atarget cell differentiated from an embryonic stem cell of the presentinvention will be explained by referring to FIG. 1 using a typicalexample.

In the first recombinant DNA, a first promoter (in the figure, CApromoter), a loxP sequence as a recombinase recognition sequence, and anEGFP gene as a selective marker for a target cell differentiated from anES cell are arranged in this order from a 5′ side, a Neo gene and a polyA signal as a marker are arranged between two loxP sequences, and thepoly A signal is arranged downstream of the EGFP gene. The markerarranged between recombinase recognition sequences is not limited to theNeo gene, but various marker genes can be used. The poly A signal isalso not particularly limited, but various poly A signals such as a polyA signal of bovine growth hormone, and a poly A signal of rabbitβ-globin can be used.

In addition, in the second recombinant DNA, a second promoter (in thefigure, Nkx2.5 gene promoter or αMHC gene promoter), and recombinase Creare arranged in this order from a 5′ side. The poly A signal is arrangeddownstream from the recombinase Cre.

When an ES cell in which the first recombinant DNA and the secondrecombinant DNA are transferred is differentiation-induced into a targetcell, thereby, a second promoter is expressed, and recombinase Cre actsto excise a part held by loxP sequences, an EGFP gene is stronglyexpressed by a first promoter, a cardiac muscular cell as a target cellcan be visualized using fluorescent light as an index, and can beselectively isolated simply and easily by flow cytometry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an explanation drawing showing schematically the method forselectively isolating or visualizing a target cell differentiated froman embryonic stem cell of the present invention.

FIG. 2 is a fluorescent micrograph image showing an efficacy of genetransfer by an adenovirus vector. (a) is a fluorescent micrograph imageof a mouse ES cell cultured on a feeder cell (R1 cell), (b) is afluorescent micrograph image of a mouse ES cell cultured without afeeder cell (D3 cell), and (c) is a fluorescent micrograph image of amouse ES cell (D3 cell) during differentiation inducement. In addition,(a) to (c) are obtained by infection with Ad.CMV-LacZ at MOI of 100, andx-gal staining, and a right upper part of each fluorescent micrographimage indicates a phase contrast micrograph image thereof.

FIG. 3 is a graph showing an efficacy of gene transfer by an adenovirusvector, and (a) is a graph showing an efficacy of gene transfer in an EScell (R1 cell) by an adenovirus vector at each MOI, and (b) is a graphshowing an efficacy of gene transfer in an ES cell (D3 cell) by anadenovirus vector at each MOI.

FIG. 4 is a fluorescent micrograph image of an ES cell which has beenvisualized with EGFP by the method for selectively isolating orvisualizing a target cell differentiated from an embryonic stem cell ofthe present invention. (a) Ad.CMV-LacZ was infected as a negativecontrol, and there is no expression of EGFP. (b) Ad.CA-LacZ was infectedas a positive control, and about 60 to 70% of cells were visualized byexpression of EGFP. (c) Ad.Nkx2.5-Cre was infected on day 4, this wasobserved on day 6, and cells appearing to be a target cell are dispersedand visualized. (d) Ad.αMHC-Cre was observed on day 13, and cellsappearing to be a target cell are visualized. FIG. 5 is a chart of flowcytometry of cells which were isolated using, as an index, expression ofAd.CMV-LacZ, Ad.Nkx2.5-Cre and Ad.αMHC-Cre.

FIG. 6 is a fluorescent micrograph image of immunological cell stainingof cells which were isolated using, as an index, expressionof (a)Ad.Nkx2.5-Cre, or (b) Ad.αMHC-Cre. (a) A left photograph image showsEGFP, and a central photograph image shows expression of a targetprotein, an upper photograph shows SMA, a lower photograph showstropomyosin (TM), and these have been obtained by immunologicalfluorescent staining with a specific antibody to each of them. A rightphotograph image is a photograph image obtained by overlaying a leftphotograph image and a central photograph image, showing that EGFP and atarget protein are expressed in the same cell. (b) A left photographimage shows EGFP, a central photograph image shows expression of atarget protein, an upper photograph shows αMHC, a lower photograph showsactinin (all cardiac muscle cell-specific molecules), and these wereobtained by immunological fluorescent staining with a specific antibodyto each of them. And, a right photograph image is a photograph imageobtained by overlaying a left photograph image and a central photographimage, showing that EGFP and a target protein are expressed in the samecell.

FIG. 7 shows an electrophoretic image of recombinase Cre expressed by aCA promoter which is a constitutive strong expression promoter, and aNkx2.5 promoter and an αMHC promoter of a tissue-specific gene.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail below by way ofexamples. Genetic technologies and cell culturing techniques forhandling plasmids, DNAs, various enzymes, Escherichia coli, and culturedcells in Comparative Examples and Examples were performed according tothe methods described in “Current Protocols in Molecular Biology, editedby F. Ausubel et al., (1994), John Wiley & Sons, Inc.” and “Culture ofAnimal Cells; A Manual of Basic Technique, edited by R. Freshney, 2ndedition (1987), Wiley-Liss” unless otherwise indicated. In addition,culturing and handling of an ES cell were performed according to themethods described in the aforementioned “Manual of Manipulation of MouseEmbryo” authored by Brigid Hogan and others, translated by KazuyaYamauchi and others, Kindai Publisher (1997), or “Gene Targetting:Preparation of Muted Mouse Using ES Cell” authored by Shinichi Aizawa,Experimental Medicine Supplement, Biomanual Series 8, Yodosha (1995)unless otherwise indicated. General handling of adenovirus was performedby the methods described in Manipulation of adenovirus vectors, Chapter11. p109-p128 authored by Frank L. Graham; Methods in Molecular Biology,Vol. 7: Gene Transfer and Expression Protocols (1991) edited by E. J.Murray unless otherwise indicated. Preparation of adenovirus wasperformed according to the methods described in Chem, S-H. et al.,Combination gene therapy for liver metastases of colon calcinoma invivo. Proc. Natl. Acad. Sci. USA. (1995) 92, 2477-2581, or Mizuguchi etal., Human Gene Ther., Vol. 9, 2577-2583, (1998).

COMPARATIVE EXAMPLE

The previous method for isolating an embryonic stem cell will bedescribed below as Comparative Example.

As a Nkx2.5 gene promoter, the promoter gifted from Mr. Yutzey was used.Yutzey et al. are studying in detail a promoter region which enablesregulation of cardiac muscle-specific expression of a Nkx2.5 gene bygenome analysis of a 5′ upstream of the Nkx2.5 gene (details aredescribed in Development. Vol. 125, 4461-4470 (1998)). This confirmedthat a region containing a transcription initiating point to −3059 bp of5′ upstream functions as an optimal promoter region for expression ofthe Nkx2.5 gene at a sufficient cardiac muscle-specific expressionlevel.

Incidently, the aforementioned article shows that even a shorter region,that is, a region of a transcription initiating point to −959 bp of 5′upstream, or conversely even a longer region, that is, a region of atranscription initiating point to −9000 bp of 5′ stream can not expressthis downstream gene at a sufficient cardiac muscle-specific expressionlevel. A pNkx2.5-IA-LacZ plasmid in which this region of a transcriptioninitiating point to −3059 bp of 5′ upstream of the Nkx2.5 gene, a lacZgene of Escherichia coli as a marker gene, SV40 small t-intron and polyA signal are inserted into a plasmid pBlueScript SK was gifted from Mr.Yutzey (details are described in the aforementioned development. Vol.125, 4461-4470 (1998)). It was confirmed that the pNkx2.5-IA-LacZ (aname described in the article is −3059Nkz2.5lacZ) expresses a LacZ genecardiac muscle cell-specifically.

Then, an EGFP (enhanced green fluorescent protein) gene was excised froma plasmid pEGFP-C1 (Clontech, catalogue No.6084-1) by treatment withrestriction enzymes NheI and BclI, and both ends of the excised EGFPgene were blunt-ended with T4 DNA polymerase I. The pNkx2.5-IA-LacZ wascut with a restriction enzyme SalI, an end was blunt-ended with T4 DNApolymerase I and, for preventing self ligation, an end wasdephosphorylation-treated with a Calf Intestine Phosphatase (CIP)enzyme, and the excised EGFP gene and T4 DNA ligase were reacted toperform a ligation reaction, to prepare a plasmid pBS-Nkx2.5-EGFP.

On the other hand, a plasmid pBS-loxP-Neo was prepared by inserting agene in which a pGK promoter, a Neo gene (G418 drug-resistant gene), anda poly A signal of bovine growth hormone were ligated in this order froma 5′ side, between HindIII-BamHI in a multicloning site between two loxPsequences of a recombinase recognition sequence of a plasmid pBS246(formally GIBCO BRL, currently Invitrogen, catalogue No.10348-019). Thisplasmid pBS-loxP-Neo was cut with a restriction enzyme NotI, CIP-treatedand purified to obtain a fragment, pBS-Nkx2.5-EGFP was cut with NotI toobtain a gene fragment (gene in which Nkx2.5 promoter of a transcriptioninitiating point to −3059, an EGFP gene, SV40 small t-intron and poly Asignal are ligated), and the both fragments were reacted with a T4 DNAligase to prepare pNkx2.5-EGFP-loxP-Neo. The pNkx2.5-EGFP-loxP-Neo is aplasmid having a backbone of pBluescript, in which a Nkx2.5 genepromoter (from transcription initiating point to −3059), an EGFP gene,SV40 small t-intron and a poly A signal, a loxP sequence, a pGKpromoter, a Neo gene, a poly A signal of bovine growth hormone, and aloxP sequence are inserted in this order from a 5′ side.

This plasmid pNkx2.5-EGFP-loxP-Neo was transferred into a R1 cell of amouse ES cell strain by an electroporation method (electricity waspassed at 150 mV and 950 μF with Gene Pulser II of Biorad in a 0.2 mmcuvette), G418 as an antibiotic for drug selection was added at aconcentration of 150 μg/ml to an ES cell medium with LIF (recombinantprotein of mouse leukemia inhibitory factor: formally GIBCO BRL,currently Invitrogen, trade name ESGRO) added from the following dayand, after 1 to 2 weeks at which a colony could be sufficientlyseparated, this drug-resistant ES cell strain clone was collected. Bythree times experiments 78 G418-resistant clones were collected.

The ES cell medium is a medium in which NaHCO₃, 125 μM 2-mercaptoethanol(Nacalai), none-essential amino acid (Invitrogen), nucleic acid, 20%fetal bovine serum (Invitrogen), streptomycin and penicillin are addedto Dulbecco's Modified Eagle's Medium (high glucose condition,L-glutamine and 110 mg/L sodium pyruvate are contained: Sigma). Uponmaintenance of undifferentiation ability of an ES cell, culturing isperformed by adding 10³ U/mL of LIF to this ES cell medium. Upondifferentiation inducement of an ES cell, culturing is performed in thisES cell medium alone without adding LIF.

A DNA was extracted from these clone ES cells, genomic PCR wasperformed, and 25 clones were positive with primers designed foramplifying EGFP, 16 clones were positive with primers designed foramplifying a region containing a ligated Nkx2.5 gene promoter and EGFP,and 13 clones were positive with a set of both primers. Since at leastthese 13 clones were thought that a target gene is correctlytransferred, these 13 double positive clones were used in an experimentthereafter.

ES cells of the 13 clones were placed on two differentiation inducementsystems. In one system, an ES cell was cultured by suspending in an EScell medium with LIF not added in a cell none-adhesive dish, thereby, acell mass similar to an initial embryo called embryoid body was formed.When this embryoid body was transferred to an adhesive culturing dishfrom 3 day (3 days after initiation of differentiation inducementwithout LIF), and cultured in the adhered state in an ES cell mediumwith LIF not added, a cell mass which was self-pulsing like a cardiacmuscle cell of a living body appeared from 7 day (7 days afterinitiation of differentiation inducement without LIF) to 14 day.

Another system is a differentiation inducement system in which an EScell is placed on a mouse stromal cell called ST2 cell, and co-culturedin an ES cell medium with LIF not added. Also in this system, when an EScell was placed on this ST2 cell without LIF and, 7 to 9 days after, acell mass which was self-shrinking like a cardiac muscle of a livingbody appeared somewhere.

When a RNA was extracted from a cell mass everyday from 7 to 14 daysafter placement on the two differentiation systems, and a RT-PCR methodwas performed, expression inducement of a gene like a cardiacmuscle-specific gene (Nkx2.5, αMHC, MLC2v etc.) was confirmed. Inaddition, expression of a muscle-specific or cardiac muscle-specificprotein (α actinin, tropomyosin, Nkx2.5, MLC2v) could be confirmed byimmunological staining of a cell mass at the same stage, and it wasdemonstrated that these self-shrinking cell masses were differentiatedinto a cardiac muscle cell.

On the other hand, even when a mRNA of an EGFP gene was amplified from aRNA extracted from a cell mass which had been differentiated into acardiac muscle at 4 days after to 14 days after by a RT-PCR method,positive finding could not be seen in any case. And, in cardiac musclecell masses differentiated from these ES cells which had been inducedfrom any ES cell clone, neither visualization with a fluorescentmicroscope, nor clear expression of EGFP at a isolatable level with acell sorter could not be seen. As described above, this Nkx2.5 genepromoter (−3059) is the same as that reported by Yutzey et al. inDevelopment. Vol. 125, 4461-4470 (1998), a gene ligated to this promoterwas expressed at a region which is scheduled to be a heart at an earlystage in development of a mouse, and it was confirmed that the gene isexpressed in a cardiac muscle cell relatively specifically.

Since the method for detecting expression of a LacZ gene by x-galstaining of a tissue used by Yutzey et al. is an enzymatic reaction,detection sensitivity is relatively high, and a level of expression of aLacZ gene necessary for detection may be very low, but in order tovisualize or isolate a cell while living, a LacZ gene cannot be used asa marker gene. For this reason, it is general to use an EGFP gene as amarker gene for such the purpose. On the other hand, in order tovisualize EGFP with a fluorescent microscope or separate it with a cellsorter, an extent of an expression amount of an EGFP gene is necessary.That is, although this gene has been correctly incorporated, and an EScell has been differentiated into a cardiac muscle, expression of EGFPis not seen because a gene ligated to this Nkx2.5 gene promoter producescardiac muscle-specific expression, but has not expression activity at alevel at which EGFP can be visualized. Like this, by the previousmethod, a cardiac muscle cell which had been differentiated into atarget cell could not be visualized by expression of an EGFP gene from aNkx2.5 gene promoter, and could not be isolated with a cell sorter,thus, both trials were impossible.

Example 1

Both of pCMV-loxP-Neo-EGFP and pCA-loxP-Neo-EGFP, a plasmid containing afirst recombinant DNA in which a CMV promoter (human cytomegalovirusimmediate early promote) or a CA promoter (cytomegalovirusenhancer+chicken β-actin promoter), a Neo gene held by two loxPsequences downstream therefrom, and an EGFP gene as a marker genefurther downstream therefrom were arranged from a 5′ side, wereprepared, and the preparation process will be described below.

First, a pcDNA3 plasmid (Invitrogen) is treated with restriction enzymesBclI and BsmI, a Neo gene is excised, and the fragment was blunt-endedby T4 DNA polymerase I treatment (insert). A pBS246 plasmid (formallyGIBCO BRL, currently Invitrogen) is cut with restriction enzymes HindIIIand BamHI, and CIP-treated (vector). Both of this insert and the vectorwere reacted with a T4 DNA ligase to perform ligation, to obtain aplasmid pBS-loxP-Neo in which a Neo gene was inserted between loxPsequences of pBS246.

Then, this pBS-loxP-Neo was excised with a restriction enzyme NotI, andtreated with T4 DNA polymerase I to recover two loxP sequences and agene fragment of a Neo gene held by them (insert). On the other hand, aplasmid pIRES-EGFP (Clontech, catalogue No.6064-7) with a CMV promotertransferred therein was treated with restriction enzymes ClaI and BamHI,a vector part from which MCS (multicloning site), IVS (syntheticintron), and IRES (internal ribosome entry site) had been removed wasrecovered, and this was blunt-ended with T4 DNA polymerase I (vector).Both of this insert and the vector were ligated with a T4 DNA ligase, toobtain one target plasmidp CMV-loxP-Neo-EGFP.

Further, the plasmid pCMV-loxP-Neo-EGFP was treated with BglII and EcoRIto remove a CMV promoter part, the fragment was blunt-ended with T4 DNApolymerase I, and CIP-treated (vector). On the other hand, a cosmidpAdexlCawt (Takara, code No.6150) was treated with PmeI and SwaI toexcise a CA promoter part, and the fragment was blunt-ended with T4 DNApolymerase I (insert) Both of this insert and the vector were reactedwith a T4 DNA ligase to perform ligation, to obtain another end plasmidpCA-loxP-Neo-EGFP.

The plasmid pCMV-loxP-Neo-EGFP or pCA-loxP-Neo-EGFP was transferred intoan ES cell (D3 cell), respectively, by an electroporation method, andthis was cultured for 1 to 2 weeks in an ES cell medium with 150 μg/mLG418 and 10³U/L LIF added thereto, to isolate an ES cell clone. Theseprocedures are as described in detail in item of Comparative Example.Then, 70 clones in which pCMV-loxP-Neo-EGFP was transferred, and 59clones in which pCA-loxP-Neo-EGFP was transferred, were collected.

Separately, a second recombinant DNA in which a Nkx2.5 promoter (−3059),a recombinase Cre gene, and a bovine growth hormone poly A signal werearranged from a 5′ side was prepared, and an adenovirus vectorAd.Nkx2.5-Cre containing the gene was further prepared as follows:

First, a plasmid pHMCMV6 (gifted from Mr. Mark Kay: details of theplasmid are described in H. Mizuguchi and M. Kay: Human Gene Ther vol.10 2013-201 (1999); currently sold by Clontech) was treated with NheIand MunI to remove a CMV promoter, and ligation was performed with a T4DNA ligase. Further, this plasmid was cut with AflII, the fragment wasblunt-ended with T4 DNA polymerase I (vector), and an insert obtained byexcising a Cre gene with XhoI and MluI from a plasmid pBS185 (formallyGIBCO BRL, currently Invitrogen), and blunt-ending this with T4 DNApolymerase I was inserted in this place by a ligation reaction with a T4DNA ligase to obtain a plasmid pHM Ap-Cre. That is, pHM Ap-Cre is aplasmid which does not have a promoter, has a multicloning site forsimply inserting an arbitrary promoter, and has a Cre gene, and a bovinegrowth hormone poly A signal downstream therefrom.

The pNXK2.5-IA-LacZ described in Comparative Example was excised withNotI and XbaI, and this was blunt-ended with a T4 DNA polymerase toobtain a Nkx2.5 gene promoter part (insert), which was ligated to avector obtained by cutting pHM Ap-Cre with NotI, blunt-ending with T4DNA polymerase I, and CIP-treating it, with a T4 DNA ligase, to obtain aplasmid pHM-Nkx2.5-Cre.

Further, an insert obtained by cutting pHM-Nkx2.5-Cre with I-CeuI andPI-SceI, and a vector obtained by cutting an adenovirus vector plasmidpAdHM4 (gifted from Mr. Mark Kay: details of the plasmid are describedin H. Mizuguchi and M. Kay: Human Gene Ther vol. 10: 2013-201 (1999))with I-CeuI and PI-ScuI were ligated with a T4 DNA ligase to obtain anadenovirus vector plasmid pAdHM4-Nkx2.5-Cre. The pAdHM4-Nkx2.5-Cre wascut with PacI and purified, which was transferred into a 293 cell, aplaque of adenovirus Ad.Nkx2.5-Cre appearing after 10 to 14 days wasrecovered, amplification of the virus was performed with a 293 cell,purified by a density gradient method with CsCl, and subjected todesulting with a column (details of the method are described in thecited reference described first).

After infection of a cell with a human 5-type adenovirus vector andtransfer into the cell, this Ad.Nkx2.5-Cre expresses a Cre gene undercontrol of a Nkx2.5 gene promoter.

In addition, according to a similar method, a CA promoter wastransferred into pHMAp-Cre and, according to a similar method, anadenovirus vector Ad.CA-Cre was prepared. The Ad.CA-Cre is a human5-type adenovirus vector and, after infection of a cell and transfer ofa gene into the cell, the vector expresses a Cre gene under control of aCA promoter. Since the CA promoter can constitutively and stronglyexpress a downstream gene in most cells including an ES cell, a cellinfected with Ad.CA-Cre expresses constitutively a Cre enzyme in thecell although in the undifferentiated state.

Then, an efficacy of transfer of a gene into an ES cell of an adenovirusvector was investigated. First, a human 5-type adenovirus vectorAd.CMV-lecZ expressing a LacZ gene under control of a CMV promoter wasprepared by the aforementioned method. When a cell was infected with theAd.CMV-LacZ at each MOI (multiplicity of infection: infectable virusnumber/cell number), and a ratio of positive cells was assessed by x-galstaining a gene transfer efficacy was increased as MOI was increased(see FIG. 2, FIG. 3). And, in an ES cell of any of R1 and D3, a genecould be transferred in many cells at MOI of 30 in the presence or inthe absence of a feeder cell, and a gene could be transferred into 100%of ES cells at MOI of 100 to 300. However, since cell disorder is seenin some cases when MOI is too extremely increased, an experimentdescribed later was performed at MOI of 30 at which a gene can betransferred into about 60 to 80% of cells while little cell disorder isseen. Similarly, when an ES cell which had been subjected to theaforementioned differentiation inducement was infected with a sufficientamount of Ad.CMV-LacZ, and this was assessed by x-gal staining, a genecould be transferred into almost cells with a slight difference at anydifferentiation stage and any day of 1 to 14 days after differentiationinducement (see FIG. 2 c) It could be confirmed that a gene can betransferred into an ES cell simply and at a high rate at anydifferentiation stage by using an adenovirus vector like this. Inparticular, since it is difficult to transfer a gene by the previousgeneral gene transferring method such as an electroporation methodduring differentiation inducement, it was seen that use of an adenovirusvector is very useful.

Among 70 clones of an ES cell in which pCMV-loxP-Neo-EGFP with a firstrecombinant DNA incorporated therein was stably transferred, first, 34was subjected to genomic PCR with a primer set designed in ComparativeExample for amplifying EGFP, and 31 among them were positive. Inaddition, when these 31 ES cell clones were infected with Ad.CA-Cre, 4clones were visualized with an expression intensity sufficient forobservation with a fluorescent microscope (all of these four clones arepositive by genomic PCR with EGFP). That is, since a clone having moreintense expression can be rapidly selected by direct screening withAd.CA-Cre infection even when insertion of an EGFP gene is not confirmedby genomic PCR, the resulting ES cell clone was selected thereafter fora target clone by expression intensity of EGFP by direct Ad.CA-Creinfection. When remaining 34 clones with pCMV-loxP-Neo-EGFP transferredstably were screened by EGFP expression intensity (by observation with afluorescent microscope) after Ad.CA-Cre infection, 11 clones were EGFPstrong expression, and 4 clones were EGFP weak expression. That is,target 15 clones (19 clones including weak expression) could becollected from a total of 70 clones with pCMV-loxP-Neo-EGFP transferredstably.

On the other hand, when 59 clones of an ES cell with pCA-loxP-Neo-EGFPtransferred stably were directly screened by EGFP expression afterAd.CA-Cre infection, 16 clones were target clones expressing EGFPstrongly.

In both of an ES cell with pCMV-loxP-Neo-EGFP transferred stably and anES cell with pCA-loxP-Neo-EGFP transferred stably, expression of EGFP ata visible level was not seen, and problematic background was not seenwhen not infected with Ad.CA-Cre, or when infected with Ad.CMV-LacZ as acontrol not expressing a Cre gene. In addition, an expression level ofEGFP of an ES cell with each of PCMV-loxP-Neo-EGFP and pCA-loxP-Neo-EGFPtransferred stably after Ad.CA-Cre infection was stronger inpCA-loxP-Neo-EGFP in any case. This seems to be due to a difference inpromoter activities that a CA promoter can induce stronger expressionthan a CMV promoter also an ES cell as reported in many cell strains.However, expression of EGFP at a visible level was obtained also in anES cell with pCMV-loxP-Neo-EGFP, and it is considered that evenpCMV-loxP-Neo-EGFP has essentially no problem in an experiment for thepurpose of analysis and isolation with a cell sorter having goodsensitivity. The following differentiation inducement experiment in thepresent experimental example was performed using pCA-loxP-Neo-EGFP froma viewpoint that expression is stronger, observation under a fluorescentmicroscope can be performed more clearly, and analysis can be performedmore easily.

Among 15 clones of an ES cell for which strong expression of EGFP isassured by stable transfer of pCA-loxP-Neo-EGFP and excision of a Neogene held by loxPs after expression of a Cre enzyme as described above,3 clones were used to perform a differentiation inducement experimentusing Ad.Nkx2.5-Cre. In a preliminary experiment using theaforementioned Ad.CA-Cre, expression of EGFP at a sufficiently visiblelevel was seen from one day after Ad.CA-Cre infection, expressionreached a maximum expression level two days after infection, and it wasconfirmed that sustained expression of EGFP at the same level isobtained thereafter.

These 3 clones of an ES cell were used in the differentiation inducementsystem via embryoid body already described in Comparative Example.First, expression of a Nkx2.5 mRNA in this differentiation inducementsystem was examined by RT-PCR everyday, and expression of Nkx2.5 wasclearly seen from 5 days after differentiation inducement. For thisreason, 4 days after differentiation inducement an ES cell duringdifferentiation was infected with Ad.Nkx2.5-Cre at MOI of 30. That is,on day 1, the cell was cultured in an ES cell medium with LIF not added,preparation of embryoid body was initiated in the none-adhered state,this was adhered on a culturing dish 3 days after initiation ofdifferentiation inducement and, on day 4, the cell was infected withAd.Nkx2.5-Cre at MOI of 30. From 5 days after initiation ofdifferentiation inducement, cells appeared somewhere for which EGFPcould be visualized under a fluorescent microscope, from 6 days afterinitiation of differentiation inducement, EGFP expression at a verystrong expression level was observed under a fluorescent microscope, andthis EGFP expression was ever seen thereafter (see FIG. 4C). On day 6 orday 8, a cell mass was dissociated with trypsin, and sufficientlyseparated into individual cells, and a target cell expressing EGFP wasisolated with a cell sorter. By analysis with this flow cytometry, apositive rate of a group infected with Ad.Nkx2.5-Cre, that is, aNkx2.5-expressing cell was about 2% (see FIG. 5). To the contrary, apositive rate of a group infected with Ad.CA-Cre instead ofAd.Nkx2.5-Cre as a positive control (that is, this represents a ratio ofa cell which was infected with adenovirus and in which a Cre gene wastransferred) was about 60% (see FIG. 5). On the other hand, a positiveratio of a group infected with Ad.CMV-LacZ instead of Ad.Nkx2.5-Cre as anegative control, or a group not infected with an adenovirus vector atall (that is, this is leakage of EGFP expression in an ES cell with agene of pCA-loxP-Neo-EGFP transferred stably in the state where loxPshave not been excised with a Cre enzyme, background) was 0 to 0.2% (seeFIG. 4 a, FIG. 5).

That is, in this experimental system, since a target gene is correctlytransferred in about 60% of cells at a very high rate, there is nobackground, and a target Nkx2.5-expressing cell can be visualized andisolated correctly, it was seen that the experimental system is an veryexcellent experimental system which has solved these techniques whichcould not be solved previously.

Then, the isolated cell was immediately seeded on culturing a cell, andcultured and, on the following day, or a few days after, nature andcharacter of the cell were analyzed. First, the isolated cell exhibitedstrong EGFP expression to an extent that expression could be clearlyconfirmed in most cells with a fluorescent microscope from on thefollowing day to a few days after. Thereby, it was confirmed that thecell isolated by this method is a target cell having a very high purity.Then, for analyzing nature and character of the cell, a RNA wasextracted from this cell, and expression of a Nkx2.5 gene, andexpression of a cardiac muscle-specific molecule, and other associatedgenes were investigated. Alternatively, these isolated cells weresubjected to immunological cell staining, and expression of thesemolecules was investigated.

First, clear expression of a Nkx2.5 gene, αMHC, MEF2c (cardiacmuscle-specific transcription factor) or GATA4 (transcription factorexpressed in a cardiac muscle at highly frequently) was seen in 80% ofcells. This result revealed that most of these cells are a cardiacmuscle cell.

On the other hand, expression of smooth muscle actin (SMA) ortropomyosin (TM) which is a marker for other than a cardiac muscle wasalso seen in 20 to 30% of cells (see FIG. 6 a). There has beenpreviously no report that only a cell expressing Nkx2.5 could beisolated assuredly (moreover, assuredly at such a level that it can bealso visualized with a fluorescent microscope) from an ES cell, and whentaken into consideration that Nkx2.5 is a transcription factor which isexpressed earliest during a process of cardiac muscle development amongcardiac muscle-specific genes known today, result that a part of thepresent cell expresses a marker for other than a cardiac muscle may leadto a possibility that the present cell is an more undifferentiated cellwhich has not previously isolated from an ES cell and is destined to bedifferentiated into a cardiac muscle cell at a differentiation stagebefore a mature cardiac muscle cell, or further a cardiac muscle whichshould be called a cardiac myoblast. For this, this isolated cell itselfis considered to be very useful particularly in future fundamental studyregarding development, differentiation or regeneration of a heart at anearly stage which has not been sufficiently clarified yet, ordevelopment of regeneration therapy in a cardiac disease using an EScell.

In addition, as various cardiac muscle-specific genes expressed at eachdifferentiation stage of a cardiac muscle, some such as MEF2C and GATA4have been reported and, from now on, a cell at each differentiationstage can be freely isolated by using the present method and usingexpression of such the gene as an index, thus, the invention of thepresent method is greatly meaningful. Further, usefulness of the presentmethod is not limited to a cardiac muscle, but by using promoters oftissue-specific or character-identified various genes, each tissue atany specific differentiation stage differentiated from an ES cell, orany target cell specialized for expression of a certain gene can bevisualized and isolated. Also from this point of view, the presentinvention has extremely important meaningfulness in regenerationmedicine or embryology using an ES cell.

Example 2

The second promoter used in the present invention is not limited to theNkx2.5 gene promoter, but can be widely used by generalization for thepurpose of visualizing and further isolating an ES cell-derived cardiaccell or ES cell-derived other cells or tissues using other cardiacmuscle-specific gene as an index under fluorescent microscope. That is,only by substituting the second promoter with an objective promoter, anyES cell-derived target cell can be visualized and isolated. In order tofurther confirm such the general wide usefulness of the presentinvention, an adenovirus vector Ad.αMHC-Cre using a mouse AMHC genepromoter as the second promoter, that is, an adenovirus vector fortransferring a recombinant gene specifically expressing a Cre enzymeinto a cardiac muscle cell in which AMHC is expressed, was prepared bythe same method as that of Example 1, and the same experiment wasperformed.

The Ad.αMHC-Cre was prepared as follows: First, about 5.5 kb of a DNAcontaining an αMHC gene promoter was excised from a plasmid with an αMHCpromoter inserted therein (details of the plasmid are described in J.Biol. Chem. Vol. 266, p9180-9185(1991)) which had been gifted from Mr.Jeffrey Robbins of University of Cincinnati, College of Medicine,blunt-ended with T4 DNA polymerase I, purified, and extracted. As Mr.Robbins et al. reported in the aforementioned Publication, this is a DNAcontaining a part of a 3′ last exon of a heart-type β myosin heavy chaingene, a 3′ region of AMHC, and first three exons not encoding a protein,and the same article shows that this region functions as a promoterresulting in heart-specific expression. On the other hand, the vectorpHM Ap-Cre prepared in Example 1 was cut with an enzyme NotI,blunt-ended with T4 DNA polymerase I, subjected to terminaldephosphorylation treatment with an enzyme CIP, and purified. This and a5.5 kb DNA fragment of the aforementioned αMHC gene promoter werereacted with a T4 DNA ligase enzyme to perform a ligation reaction, toprepare a plasmid pHM-α MHC-Cre in which a Cre gene is ligated todownstream of the αMHC gene promoter. This pHM-αMHC-Cre and theadenovirus vector plasmid pAdHM4 were cut with restriction enzymesI-CeuI and PI-SceI as described in Example 1, and both plasmids wereligated with a T4 DNA ligase to obtain pAdHM4-αMHC-Cre. ThepAdHM4-αMHC-Cre was cut and purified, which was transferred in a 293cell, a plaque of the adenovirus Ad.αMHC-Cre appearing 10 to 14 daysafter was recovered, and the virus was amplified, purified, and desaltedas described in Example 1. The thus prepared Ad.αMHC-Cre expresses a Cregene under control of the AMHC gene promoter after infection of a celland transfer of a gene into the cell.

Among the ES cell clone strains in which the pCA-loxP-Neo-EGFP preparedExample 1 was stably transferred, the ES cell clone strain for whichstrong expression of EGFP after expression of Cre was confirmed was usedto perform the same experiment as that of Example 1 employingAd.αMHC-Cre in place of Ad.Nkh2.5-Cre. The fundamental experimentalprotocol and procedure were the same as those described in Example 1,provided that in Example 1, a cell was infected with Ad.Nkx2.5-Cre 4days after differentiation inducement, and the visualized target cellwas isolated with a cell sorter on day 6, while in the present Example,a cell was infected with Ad.1αMHC-Cre on day 9, and a visualized targetcell was isolated with a cell sorter on day 13. The reason is asfollows: When expression of an endgenous αMHC gene in an ES cell afterinitiation of differentiation inducement was investigated by a RT-PCRmethod and immunological histochemistry, expression of αMHC wasremarkably perceived from about day 8 to day 14. The result of a time ofexpression of this αMHC in differentiation of an ES cell is consistentwith the fact in a living body that since αMHC is one of cardiacmuscle-specific shrinkage proteins, strong expression is recognized in amature cardiac muscle cell, and is also consistent with the fact that acolony of an ES cell exhibiting cardiac muscle-like pulsing is mostremarkably recognized on day 9 to 14 after differentiation inducement.Like this, according to the same manner as that of Example 1 except thata time of infection with Ad.αMHC-Cre, the following experiment wasperformed.

From day 1 after infection with Ad.αMHC-Cre (day 9 after differentiationinducement), expression of EGFP was recognized in a part of cells undera fluorescent microscope, from day 2 after infection (day 10 afterdifferentiation inducement), expression became clear, thereafter,expression was slightly enhanced and, on day 4 after infection (day 13after differentiation inducement), expression of EGFP became maximum.Therefore, at this term point, cells were dissociated with trypsin, andEGFP-positive cells were isolated with a cell sorter (see FIG. 4 d).Most of the isolated cells were expressing EGFP. Further, in order toconfirm property of these cells, a mRNA of a cardiac muscle-specificmolecule such as αMHC and actinin, and expression of a protein wereinvestigated by RT-PCR and immunological staining, and expression ofthese cardiac muscle-specific molecules was positive in most of theisolated cells (see FIG. 6 b). Further, a cell structure peculiar in acardiac muscle cell such as a striated muscle fiber structure could beconfirmed by observation with an electron microscope. Inter alia,observation of cardiac muscle-like pulsing of a cell for a few days fromthe next day after culturing while a cell was contacted with a culturingdish showed that the isolated cell is a target matured cardiac musclecell. From these results, it was confirmed that the isolated cell is amatured cardiac muscle cell. Like this, since a cardiac muscle cell at arelatively unmatured differentiation stage could be assuredly isolatedin Example 1, and a matured cardiac muscle cell could be assuredlyisolated in Example 2 using promoters of two genes having such thedifferent characters that Nkx2.5 is a transcription factor and αMHC is ashrinkage protein, by the present method, it was confirmed that thepresent invention is widely used by generalization, and is useful.

Example 3

While a method of taking first an ES cell clone in which the firstrecombinant DNA could be stably transferred, and transferring the secondrecombinant DNA into the ES cell by an adenovirus vector during aprocess of differentiation inducement was used in Example 1, the firstrecombinant DNA and the second recombinant DNA were directly transferredin an ES cell using an adenovirus vector during a process ofdifferentiation inducement, without the work of taking an ES cell clonein Example 3.

For doing so, first, an adenovirus vector into which the firstrecombinant DNA can be transferred, was prepared as follows:

First, a target DNA fragment in which a CA promoter, a neo gene held byloxP sequences of a recombinase recognition sequence, a poly A sequence,an EGFP gene, and a poly A sequence were ligated from a 5′ side wasexcised from the pCA-loxP-Neo-EGFP and the pCMV-loxP-Neo-EGFP preparedin Example 1 by SalI enzyme treatment. On the other hand, a SalIrecognition sequence of a multicloning site of a pHM5 plasmid (giftedfrom Mr. Mark Kay: details of the plasmid are described in H. Mizuguchiand M. Kay. Human Gene Ther vol. 10:2013-2017(1999); having restrictionenzyme recognition sequences of 1-CeuI and PI-SceI, on both ends of amulticloning site, respectively) of a shuttle vector for preparingadenovirus was cut with a SalI enzyme, subjected to terminaldephosphorylation treatment with a CIP enzyme, and this and theaforementioned excised target DNA fragment were ligated with a T4 DNAligase enzyme to obtain shuttle vector plasmids pHM-CA-loxP-Neo-EGFP andpHM-pCMV loxP-Neo-EGFP in which the objective first recombinant DNA wasinserted, respectively. Further, a target gene part was excised from thepHM-CA-loxP-Neo-EGFP or the pHM-pCMV-loxP-Neo-EGFP with an enzyme I-CeuIor PI-SceI, and an adenovirus vector plasmid pAdHM4 treated withenzymes-CeuI and PI-SceI was ligated with a T4 DNA ligase as alsodescribed in Example 1 to obtain adenovirus vector plasmidspAdHM4-CA-loxP-Neo-EGFP and pAdFM4-pCMV-loxP-Neo-EGFP having theobjective first recombinant DNA, respectively.

Preparation of an adenovirus vector was performed as described inExample 1. That is, the pAdHM4-CA-loxP-Neo-EGFP or thepHdHM4-pCMV-loxP-Neo-EGFP was treated with a PacI enzyme, transfectedinto a 293 cell, and the resulting virus plaque was amplified, purifiedand desalted to obtain an adenovirus vector Ad.CA-loxP-Neo-EGFP orAd.CMV-loxP-Neo-EGFP containing the objective first recombinant DNA.

As described also in Example 1, there is no essential difference in a CApromoter and a CMV promoter in an object of the present Example, butresults of an experiment using Ad.CA-loxP-Neo-EGFP are shown below dueto stronger expression. In this respect, the same experiment wasperformed using Ad.CMV-loxP-Neo-EGFP, and it was confirmed that the sameresults are obtained.

Using an ES cell with no gene transferred (D3), differentiationinducement was performed by preparing embryoid body in a mediumexcluding LIF as in Example 1.

First, for isolating an more undifferentiated cardiac muscle cellderived from an ES cell using Nkx2.5 as an index, two adenovirus vectorsof Ad.Nkx2.5-Cre and Ad.CA-loxP-Neo-EGFP were infected at MOI of 30 onday 4, and a target EGFP-expressing cell was isolated with a cell sorteron day 6. That is, the present Example is different from Example 1 onlyin that the first recombinant DNA was transferred using an adenovirusvector. As a result of this experiment, the same cells as those ofExample 1 were isolated, and these cells exhibited the same geneexpression pattern as that of Example 1.

Then, for isolating a mature cardiac muscle cell derived from an ES cellexpressing an αMHC gene, two adenovirus vectors of Ad.αMHC-Cre andAd.CA-loxP-Neo-EGFP were infected at MOI of 30, respectively, on day 9after differentiation inducement as in Example 2, and a targetEGFP-expressing cell was isolated with a cell sorter on day 13. Thisexperimental result also showed that the same cells as those of theexperiment 2 were isolated, and these cells exhibited a gene expressionpattern and cardiac muscle cell-like shrinkage characteristic in acardiac muscle similarly.

From results of these two experiments, it was confirmed that, bytransferring the first recombinant DNA or the second recombinant DNAusing adenovirus, the same result as that of the previous method oftaking a stably expressing cell with a gene transferred therein can beobtained more simply.

A method of preparing an ES cell stably expressing the first recombinantDNA by the prior art, selecting a better clone, and transferring thesecond recombinant DNA into the clone with adenovirus, as shown inExample 1 and Example 2, and a method of transferring both DNAs usingadenovirus as in Example 3 have no essential difference inmeaningfulness in the present invention and, on the other hand, it isthought that it is useful to use respective advantages of the twomethods depending on an object.

Advantages of using an adenovirus vector for transferring two DNAs aresummarized as follows:

(1) The first and second recombinant DNAs are stably transferred, andlabor and a time of troublesome work of taking a constitutivelyexpressing clone are not necessary: That is, as shown in Examples 1 and2, in order to transfer the first recombinant DNA by the prior art, andselectively take a stably and constitutively expressing clone, labor anda time are necessary. Further, if transfer of a gene is performed onlyby the prior art without using no adenovirus vector, and a clone stablyand constitutively expressing both of the first and second recombinantDNAs is selectively taken, a further time and further labor arenecessary. In this case, besides, two kinds of different drug-resistantgenes are necessary, there is a possibility that troublesome work ofexpression of these multiple drug-resistant genes, and selection of theclone, and influence of long term drug use on an Es cell, that is,change in character of a cell become problematic, and a target clone cannot be taken due to such the various influence in some cases.

(2) An adenovirus vector can transfer a target gene simply and at a highrate at an arbitrary time of a differentiation stage: This wasimpossible by the prior art, but the present invention has shown thatthis can be simply done using adenovirus. Advantage from this isassociated with the description of (1) and is that unnecessary influencesuch as unnecessary long term exposure to a gene and a drug is not givento an ES cell.

(3) Since an adenovirus vector stably expresses a gene transferred intoa host cell (in this case, ES cell) as an episomal form (presence in anucleus without incorporation into a chromosome) for a long term, stableresult is obtained: In the case of a method of taking an ES cell clonein which a target gene is incorporated into a chromosome utilizing adrug-resistant gene by the previous method, since the transferred geneis randomly incorporated into a chromosome, there is influence of aplace where a gene is incorporated on a chromosome, or influence of achromatin structure. For this reason, since all incorporated genes arenot necessarily expressed stably, a time and labor are necessary forselecting a better clone stably expressing an transferred gene asdescribed in (1). Further, in the case of an ES cell, it is known thatexpression of a gene incorporated into a chromosome easily becomesunstable, for example, expression of an transferred gene is shut off insome cases, as compared with the case using other cancer cell strain ora primary normal cultured cell. To the contrary, when a gene istransferred with an adenovirus vector, there is hardly such theinfluence of a chromosome or chromatin due to episomal, stableexpression is obtained, and reproducible and stable result is alwaysobtained.

(4) An arbitrary ES cell strain can be used: Differentiating ability andcharacter are different depending on a kind of an ES cell strain, and aclone and a subclone thereof. In this case, for isolating a certaincell, there is contemplated the case where a clone of an ES cell strainwhich has been identified that the clone is more easily differentiatedinto the cell is intended to be particularly used. By using anadenovirus vector which can transfer the first and second recombinantDNAs of Example 3, it is not necessary to prepare respective ES cellstrains and stable cell strains for a clone, and the cell can beisolated by freely using a desired ES cell.

(5) Since a gene can be transferred simply into cells other than an EScell, specificity of the prepared first recombinant DNA can be confirmeddirectly in other cells. In addition, construction of this gene can besimply applied to an experiment on other cells.

Example 4

As described above, specificity of the Nkx2.5 gene promoter and the αMHCgene promoter used in Examples has been already confirmed, and it wasmade clear in the actual experimental results that a cardiac muscle cellcan be isolated. Further, from a viewpoint of the (5), for the purposeof directly demonstrating that a DNA transferred with two adenovirusesof Example 3 can specifically visualize a target cardiac muscle cellcorrectly, a mouse primary cardiac muscle cultured cell was infectedwith these two adenoviruses.

A cardiac muscle was taken out from a neonatal mouse on day 1 afterbirth, and this was digested with collagenase to separate a cardiacmuscle cell into a single cell, and was seeded on a culturing dish,followed by culturing.

This procedure of culturing a primary cardiac muscle cell wasfundamentally performed according to the method described in Khalid M Aet al. Circ. Res. 72, p725-736(1993), and Wang L et al. Circ. Res. 79,p79-85(1996). The cardiac muscle cell on day 2 after such the culturingwas infected with Ad.Nkx2.5-Cre and Ad.CA-loxP-Neo-EGFP, respectively,at MOI of 5, this was observed under a fluorescent microscope 72 hoursafter infection, and the cardiac muscle cell with a gene transferredtherein was visualized as EGFP-positive. When the cardiac muscle cellwas infected with Ad.αMHC-Cre and Ad.CA-loxP-Neo-EGFP similarlyaccording to the same protocol, the cardiac muscle cell with a genetransferred therein was visualized as EGFP-positive. Other several kindsof cultured cells (Hela human uterus cervical cancer cell, MKN28 humanstomach cancer cell, LL2 mouse lung cancer cell, LM8 mouse bone sarcomacell etc.) other than a cardiac muscle were infected with these twoadenovirus vectors, and specificity thereof was tested, but such theexpression of EGFP was not seen with a fluorescent microscope in allcells. It was directly demonstrated that, when the first recombinant DNAand the second recombinant DNA used in Examples 1, 2 and 3 aretransferred, they clearly target only a cardiac muscle cell andvisualize it with EGFP like this.

In addition, an experiment was performed in order to investigatepromoter activity of a CA promoter as a constitutive strong expressionpromoter, and a Nkx2.5 promoter and an αMHC promoter of atissue-specific gene. That is, Western blotting analysis (Cre antibody)was performed using a mouse primary cultured cardiac muscle cell and aNIH3T3 mouse fibroblast strain infected with the following adenovirusvectors at an infection efficacy (MOI) of 30 or 500 (see FIG. 7). CA wasinfected with Ad.CA-Cre, and 5 μg of a protein was electrophoresed, CA′was infected with Ad.CA-Cre, and 0.5 μg of a protein waselectrophoresed, CA″ was infected with Ad.CA-Cre, and 0.1 g of a proteinwas electrophoresed, NC was a negative control not infected with Ad, and5 μg of a protein was electrophoresed, Nkx2.5 was infected withAd.Nkx2.5-Cre, and 5 μg of a protein was electrophoresed, αMHC wasinfected with Ad.αMHC-Cre, and 5 μg of a protein was electrophoresed,and Tublin (protein secreted in a cardiac muscle cell or a fibroblast)was an endgeneous control, and was detected using an anti-Tublinantibody. From FIG. 7, regarding a CA promoter, recombinase Cre wasdetected in 10-fold diluted CA′ and 50-fold diluted CA″ in addition tothe case where 5 μg of a protein was used and, regarding both of Nkx2.5promoter and αMHC promoter, a very small amount was only detected in 5μg of any protein. From this, it can be seen that promoter activity of aNkx2.5 promoter and an αMHC promoter of a tissue-specific gene is veryweak, and a selective marker such as EGFP is expressed only by thepresent invention.

INDUSTRIAL FIELD OF APPLICABILITY

The method for selectively isolating or visualizing a target celldifferentiated from an embryonic stem cell is not only widely useful inembryology, regeneration medicine and other molecular biology study byusing various isolated cells and tissues, but also very useful indevelopment of future regeneration medicine of various inveteratediseases including cardiac infarct and cerebral infarction. In addition,since a cell which has been further differentiated from a permanentlyand constitutively differentiated target cell can be also labeled andvisualized, it becomes possible to observe and analysis a differentiatedline and a tissue line on a culturing dish. Selective isolation orvisualization of a target cell differentiated from an embryonic stemcell can be performed further simply by using a kit for isolation orvisualization.

The present invention is not limited to the aforementioned Examples asfar as included in the substantial scope of the invention.

1. A method for selectively isolating or visualizing a target celldifferentiated from an embryonic stem cell, which comprises transferringa first recombinant DNA in which a first promoter, a gene havingrecombinase recognition sequences on both ends, and a selective markergene of a target cell differentiated from an embryonic stem cell arearranged in this order from a 5′ side, and the first promoter makes theselective marker gene express, and a second recombinant DNA in which asecond promoter specifically expressing in a target cell differentiatedfrom an embryonic stem cell, and a recombinase-expressing gene arearranged in this order from a 5′ side, respectively, into an embryonicstem cell.
 2. The method for selectively isolating or visualizing atarget cell differentiated from an embryonic stem cell according toclaim 1, wherein the recombinase recognition sequence is loxP.
 3. Themethod for selectively isolating or visualizing a target celldifferentiated from an embryonic stem cell according to claim 1, wherethe first promoter is a constitutive strong expression promoter.
 4. Themethod for selectively isolating or visualizing a target celldifferentiated from an embryonic stem cell according to claim 3, whereinthe constitutive strong expression promoter is a CMV promoter or a CApromoter.
 5. The method for selectively isolating or visualizing atarget cell differentiated from an embryonic stem cell according toclaim 1, wherein the selective marker gene is a fluorescent proteingene.
 6. The method for selectively isolating or visualizing a targetcell differentiated from a embryonic stem cell according to claim 1,wherein the recombinase-expressing gene is a recombinase Cre-expressinggene.
 7. The method for selectively isolating or visualizing a targetcell differentiated from an embryonic stem cell according to claim 1,wherin the second promoter is a Nkx2.5 gene promoter or an αMHC genepromoter.
 8. The method for selectively isolating or visualizing atarget cell differentiated from an embryonic stem cell according toclaim 1, wherein transfer of the first recombinant DNA into an embryonicstem cell is performed using a first vector for transferring a gene. 9.The method for selectively isolating or visualizing a target celldifferentiated from an embryonic stem cell according to claim 8, whereinthe first vector for transferring a gene is a virus vector.
 10. Themethod for selectively isolating or visualizing a target celldifferentiated from an embryonic stem cell according to claim 9, whereinthe virus vector is an adenovirus vector.
 11. The method for selectivelyisolating or visualizing a target cell differentiated from an embryonicstem cell according to claim 1, wherein transfer of the secondrecombinant DNA into an embryonic stem cell is performed using a secondvector for transferring a gene.
 12. The method for selectively isolatingor visualizing a target cell differentiated from an embryonic stem cellaccording to claim 11, wherein the second vector for intruding a gene isa virus vector.
 13. The method for selectively isolating or visualizinga target cell differentiated from an embryonic stem cell according toclaim 12, wherein the virus vector is an adenovirus vector.
 14. Anembryonic stem cell in which the first recombinant DNA as defined inclaim 1 is transferred.
 15. The embryonic stem cell in which the secondrecombinant DNA as defined in claim 1 is transferred.
 16. The embryonicstem cell in which the first recombinant DNA and the second recombinantDNA as defined in claim 1 are transferred, respectively.
 17. Theembryonic stem cell according to any one of claim 14 to claim 16,wherein the embryonic stem cell is derived from a mouse.
 18. A firstvector for transferring a gene, which comprises the first recombinantDNA as defined in claim
 8. 19. The first vector for transferring a geneaccording to claim 18, which is a virus vector.
 20. The first vector fortransferring a gene according to claim 19, wherein the virus vector isan adenovirus vector.
 21. A second vector for transferring a gene, whichcomprises the second recombinant DNA as defined in claim
 11. 22. Thesecond vector for transferring a vector according to claim 21, which isa virus vector.
 23. The second vector for transferring a gene accordingto claim 22, wherein the virus vector is an adenovirus vector.
 24. A kitfor isolation or visualization used in a method for selectivelyisolating or visualizing a target cell differentiated from an embryonicstem cell, which comprises the first vector for transferring a gene asdefined in claim 18, and the second vector for transferring a gene asdefined in claim
 21. 25. The kit for isolation or visualization used ina method for selectively isolating or visualizing a target celldifferentiated from an embryonic stem cell according to claim 24,wherein the first vector for transferring a gene and the second vectorfor transferring a gene are a virus vector.
 26. The kit for isolation orvisualization used in a method for selectively isolating or visualizinga target cell differentiated from an embryonic stem cell according toclaim 25, wherein the virus vector is an adenovirus vector.
 27. The kitfor isolation of visualization used in a method for selectivelyisolating or visualizing a target cell differentiated from an embryonicstem cell, which comprises the embryonic stem cell as defined in claim14, and the second vector for transferring a gene as defined in claim21.
 28. The kit for isolation or visualization used in a method forselectively isolating of visualizing a target cell differentiated froman embryonic stem cell according to claim 27, wherein the second vectorfor transferring a gene is a virus vector.
 29. The kit for isolation orvisualization used in a method for selectively isolating or visualizinga target cell differentiated from an embryonic stem cell according toclaim 28, wherein the virus vector is an adenovirus vector.
 30. The kitfor isolation or visualization used in a method for selectivelyisolating or visualizing a target cell differentiated from an embryonicstem cell, which comprises the first vector for transferring a gene asdefined in claim 18, and the embryonic stem cell as defined in claim 15.31. The kit for isolation or visualization used in a method forselectively isolating or visualizing a target cell differentiated froman embryonic stem cell according to claim 30, wherein the first vectorfor transferring a gene is a virus vector.
 32. The kid for isolation orvisualization used in a method for selectively isolating or visualizinga target cell differentiated from an embryonic stem cell according toclaim 31, wherein the virus vector is an adenovirus vector.
 33. A cellobtained by the method for selectively isolating or visualizing a targetcell differentiated from an embryonic stem cell as defined in claim 1.34. The cell according to claim 33, wherein the cell is a cell obtainedby using a Nkx2.5 gene promoter as the second promoter.
 35. The cellaccording to claim 33, wherein the cell is a cardiac muscle cellobtained by using an αMHC gene promoter as the second promoter.
 36. Atissue, which comprises the cell as defined in claim
 33. 37. A methodfor treating a disease, which comprises using the cell as defined inclaim 33 and/or the tissue as defined in claim
 36. 38. The method fortreating a disease according to claim 37, wherein the disease is acardiac disease.